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Mechanisms of Improving Collateral Blood Flow During Ischemic Stroke
Undergraduate Honors Research Thesis
Presented in Partial Fulfillment of the Requirements for graduation “with Honors Research Distinction” in the undergraduate School of Arts and Science of
The Ohio State University
By: Seth Teplitsky
The Ohio State University
April 2014
Project Advisor: Professor Dr. Cameron Rink, Department of Surgery
Thesis Committee: Dr. Cameron Rink, Ph.D, Advisor
Dr. Savita Khanna, Ph.D Dr. Ciaran Powers, Ph.D, MD
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Abstract:
More than 795,000 Americans suffer a stroke annually, and it is the third leading
cause of death in the United States1. There are two types of stroke, hemorrhagic and
ischemic. Hemorrhagic stroke is caused by the rupture of blood vessels within the brain,
as opposed to ischemic strokes, which are caused by a blockage within the vessel. Of
all strokes presented clinically, 86% are ischemic by nature1. Currently, there are few
treatments for ischemic stroke. The prevailing early treatments include thrombolytic
therapy and antiplatelet therapy such as low dose aspirin2,3. These treatments are each
flawed. Tissue plasminogen activator (tPA) is currently the only FDA-approved
thrombolytic therapy for the acute treatment of ischemic stroke4. This treatment is
approved for less than 10% of patients, and is given to less than 4%4. In addition,
greater than 65% of hospitals in America have never administered tPA to patients due
to low efficacy as well as potential harmful side effects5. Antiplatelets, such as low dose
aspirin, are another treatment option. These are drugs, and therefore have negative
side effects associated with long-term use such as increased risk of hemorrhagic
stroke6, and gastrointestinal bleeding7. As a result, there is a distinct lack of safe
therapeutic options for both the early and long-term treatment of ischemic stroke
patients.
Cerebrovascular collaterals refer to the network of blood vessels that are
clinically documented to perfuse stroke-affected tissue during ischemic stroke and
reduce brain injury8. While strategies to improve collateral blood flow during stroke are
of significant therapeutic interest, mechanisms and a means to improve circulation
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through these blood vessels during stroke remain unknown. This honors thesis proposal
rests on a key in vivo observation that supplementation of a lesser-characterized natural
vitamin E, alpha-tocotrienol (TCT), improves cerebrovascular collateral blood flow and
attenuates stroke injury8. TCT therefore serves as a powerful tool to study
cerebrovascular collateral remodeling during stroke. The overall objective of this honors
thesis will be to characterize the effects of TCT on cerebrovascular collateral perfusion
during stroke and to identify a mechanistic basis for TCT improvement of
cerebrovascular collateral circulation. Many previously identified arteriogenic markers,
including Tissue inhibitor of metalloproteinase 1 (TIMP1), will be investigated as a
known molecular target of interest for induction of collateral growth in the brain. FITC-
lectin tagging of cerebrovascular collaterals will be used for laser capture
microdissection experiments and downstream molecular study of arteriogenic targets.
This approach will enable the specific collection of perfused cerebrovascular collaterals
from stroke-affected tissue for mechanistic study.
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Acknowledgements I would like to sincerely thank my advisor Dr. Cameron Rink. He has been an
exceptional mentor, thoroughly peaking my interest in research, and aided me through
every step of the process leading to the completion of this project, for which I am
extremely grateful. Additionally, I would like to thank all the lab members who have
helped train me along the way, and provided the necessary resources along the way.
Specifically, I would like to recognize Kevin Olickal and Mallory Hiegel, for their
important contributions to this project. Lastly, I would like to express my gratitude to the
other committee members, Dr. Savita Khanna and Dr. Ciaran Powers. The time and
energy put in to helping me complete this thesis was greatly appreciated and not
unnoticed.
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Table of Contents
Abstract............................................................................................................................2
Acknowledgments........................................................................................................... 4
List of Tables........................................................................................................ 6
List of Figures....................................................................................................... 7
Chapter 1: Introduction.................................................................................................... 9
Chapter 2: Methodology................................................................................................ 20
Chapter 3: Results......................................................................................................... 35
Chapter 4: Discussion………………….…….................................................................. 66
References Cited .......................................................................................................... 79
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List of Tables:
1. Primary literature reviews on arteriogenic factors
2. Vitamin E Extraction Table
3. Laser Capture Microdissection Collection Yield
4. Primer Sequences
5. RNA Yield and Purity from NanoDrop
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List of Figures:
1. Chemical structure of α-tocopherol and α-tocotrienol
2. Ischemic stroke publications over time
3. Arteriogenesis publications over time
4. Tocotrienol publications over time
5. Vitamin E supplementation of mice via oral gavage
6. Surgical model for ischemic stroke in mice
7. HPLC Data Acquisition Method
8. HPLC Chromatograph
9. Embedding mouse brain for downstream sectioning and laser capture
microdissection
10. Laser Capture Microdissection of FITC-lectin perfused vessels in the brain
11. Experimental timeline
12. Oral alpha-tocotrienol (TCT) is delivered to brain tissue and attenuates
acute ischemic stroke-induced lesion volume
13. Oral TCT improves perfusion in stroke-affected MCA territory
14. Protein quantification of brain tissue
15. Oral alpha-tocotrienol (TCT) is delivered to brain tissue
16. Collateral blood flow of anastomosing vessels in the brain
17. Immunohistochemical staining of brain tissue for endothelial quantification
18. Expression of cell specific markers in laser captured collaterals
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19. MMP2 mRNA expression from laser captured collaterals
20. MMP9 mRNA expression from laser captured collaterals
21. TIMP1 mRNA expression from laser captured collaterals
22. TIMP2 mRNA expression from laser captured collaterals
23. CLIC1 mRNA expression from laser captured collaterals
24. CLIC4 mRNA expression from laser captured collaterals
25. KLF2 mRNA expression from laser captured collaterals
26. RT-PCR amplification plot of CD44v3
27. PCAF mRNA expression from laser captured collaterals
28. EphrinB2 mRNA expression from laser captured collaterals
29. Delta-Like 1 mRNA expression from laser captured collaterals
30. Delta-Like 4 mRNA expression from laser captured collaterals
31. Schematic representation of arteriogenesis and the studied markers
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Introduction:
Vitamin E: Discovery and Characterization. Vitamin E was discovered in 1922 by
Herbert Evans9 when he identified phytochemical factors essential for rodent
reproduction. This work was later continued by Barnett Sure who extrapolated its
importance to humans, and named it “vitamin E” according to the micronutrient naming
conventions of the time10. The chemical name assigned for the molecule was
tocopherol, stemming from the Greek term for childbirth, which is “tocos”11. Vitamin E’s
importance was not fully understood until more modern technology and theory of the
1950’s allowed for its elucidation in the context of the developing field of redox biology.
Palm oil provides the greatest source of a lesser-characterized vitamin E isomer, α-
tocotrienol, which has antioxidant effects as well as antioxidant-independent health
benefits shown to affect a broad range of pathological disorders11. Crude palm oil
contains up to 800 mg/kg by weight of the α-tocotrienol isomer12. Palm oil has been
documented in human diet as far back as 3000 BC9, but did not become prevalent in
diet until the early 20th century. The palm oil comes from the fruits of the oil palm tree,
Elaeis guineensis12. The oil accounts for about 30% of the world’s total oil production13,
with the most recent annual output of 58.2 million tons14. This oil is primarily produced in
Southeast Asia15, specifically Malaysia and Indonesia, and is still relatively scarce in
western diet despite its growing prominence and production on a global scale. This is
likely due to the oils higher content of saturated fatty acids (SFA) when compared to
other common vegetable oils16, which is assumed to cause an increase in low density
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lipoprotein (LDL) cholesterol as increased SFA are known to do17. There is however
conflicting scientific evidence of this outcome from oil-rich diets18,19.
Today vitamin E is known to consist of eight isomers divided into two families,
tocopherols as discovered by Evans, and tocotrienols as discovered by Pennock and
Whittle in 196320. Tocopherols consist of a chromanol ring and a 15-carbon saturated
tail derived from homogentisate (HGA) and phytyl diphosphate, respectively21. The
tocotrienols possess the same chromanol head structure, varying only in the carbon tail
where they contain 3 trans doubles bonds at positions 3’, 7’, and 11’. Both compound
families contain α, β, ɣ, and δ isoforms, differing in position and degree of methylation
on the chromanol head of the compound (Figure 1). In addition to structural differences,
a growing body of scientific literature11,21 supports independent biological functions for
vitamin E family members, some of which are known to be independent of its classically
defined antioxidant function.
Key differences between tocopherol and tocotrienol family members. When the
two families are compared, their prevalence in the plant kingdom is unequal.
Tocopherols as compared to tocotrienols are much more abundant in nature22-24. The
tocopherols are mainly found in the leaves and seeds of most dicots, and are present as
the prominent vitamin E component, whereas the tocotrienols are mainly found in the
seeds of most monocots and a limited number of dicots25. More detailed analysis has
been done to show that tocotrienols are mainly focused in non-photosynthetic tissue25.
In light of the unique biological distribution of vitamin E family members, growing
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evidence supports unique biological function for tocopherols and tocotrienols as well.
The research that has focused on the lesser-characterized forms of Vitamin E lends
support to unique biological function compared to α-tocopherol. Such biological
functions seen in tocotrienols but not tocopherols include inhibition of HMG-CoA
Reductase, the same enzyme targeted by the statin class of drugs that is responsible
for cholesterol synthesis26,27. Tocotrienol vitamin E has also been reported to inhibit
growth of human breast cancer28, and protect neurons from ischemic stroke induced
brain injury29. The study of these lesser-characterized forms of vitamin E remained
relatively untouched until the past decade. While the most bioavailable form of vitamin
E, α-tocopherol, is well studied and has a known selective transport system in
mammals, little remains known about tocotrienol uptake and transport. It is estimated
that only 1% of all the vitamin E research produced in the last 30 years pertains to
tocotrienols11. The current work aims to further the findings of unique, anti-oxidant
independent functions within the lesser-studied vitamin E isomers.
Research efforts related to ischemic stroke and the process of arteriogenesis have
increased dramatically over the past twenty-five years. A search of available literature
using “ischemic stroke” as a key term in PubMed revealed 23,302 publications in the
quarter century period of 1980–2004. By comparison, in the past decade alone there
have been 34,268 articles published (2005 through February 2014, Figure 2). During
the same time periods, a PubMed search using arteriogenesis as the key word revealed
only 202 publications in the twenty-five year period spanning 1980–2004. In the past
decade, however, there have been 544 published (Figure 3). A similar trend was
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observed using tocotrienol as a keyword (Figure 4). Taken together, the dramatic
increase in publication efforts across these distinct disciplines suggests growing clinical
and patient awareness. As it specifically relates to arteriogenesis, primary literature
addressing current molecular targets for arteriogenesis has been reviewed for the
purpose of screening TCT sensitive arteriogenic factors (Table 1). The summarizing
table is organized by target, and briefly describes their known role in arteriogenic
remodeling of collaterals.
Tocotrienol and Neuroprotection. The biological significance of vitamin E was
developed on the basis of its antioxidant function as the body’s primary lipid soluble
defense mechanism against oxidative stress and injury30. As the vitamin E family grew
with the identification of new naturally occurring isoforms (i.e. tocotrienols), so did
research efforts elucidating antioxidant independent biological function. For example, a
recent and growing body of research on the lesser-characterized isomer, alpha-
tocotrienol, has identified neuroprotective properties against both glutamate and stroke
induced neurodegeneration at nanomolar quantities. Importantly, these properties are
unique to alpha-tocotrienol, as the better-characterized alpha-tocopherol does not
possess these effects in the nanomolar concentration range29. Any biological activity at
this concentration cannot be attributed to antioxidant function, as other lipid soluble
antioxidants are present in brain tissue at much higher quantity11. This observation
points to specific mechanisms of biological function for vitamin E family members that
are unrelated to their well-known role as antioxidants.
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Multimodal Tocotrienol Protection Against Ischemic Stroke. In light of the failure of
neuroprotective agents in rodent stroke models to translate to clinical success, the TCT-
dependent protection observed in rodent models had to be tested in a large animal
setting before going to patient trials. Outcomes of a large animal (canine) trial validated
prophylactic supplementation of TCT for protection of grey and white matter brain tissue
against acute ischemic stroke31. The study demonstrated a significant decrease in post-
stroke infarct size from those animals supplemented with TCT as opposed to the
placebo control31. Furthermore, an unexpected finding was uncovered in the canine
study that was enabled by real-time angiography of cerebral blood flow. Prior to this
work, cerebral angiography was not investigated in rodent stroke models testing TCT.
Strikingly, retrospective review of cerebral angiograms identified improved collateral
blood flow in stroke-affected tissue of TCT supplemented canines as compared to
placebo controls. Quantitative analyses of collateral blood flow was determined from
cerebral angiograms using an 11-point clinical scale. Of interest, stroke-induced lesion
volume correlated tightly with collateral score, such that the canines supplemented with
TCT had greater collateral blood flow and smaller stroke lesion volumes31.
From here, the current work moves back to small animal models in order to find the
mechanistic basis for how TCT improves cerebrovascular collateral blood flow and
attenuates stroke injury. It is hypothesized that the collateral blood flow improvement
found from supplementation of α-tocotrienol is a results of arteriogenesis. Arteriogenesis
refers to the remodeling of pre-existing collateral blood vessels, whereas angiogenesis
is the growth of new collateral blood vessels altogether. To study the mechanistic basis
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of improved collateral blood flow via arteriogenesis, previously identified arteriogenic
factors including tissue inhibitor of metalloproteinase 1 (TIMP1), and matrix
metalloproteinase 9 (MMP9) were studied as known molecular targets of interest32,33.
TIMP1 plays the role of inhibitor to the MMP family. The MMP family of peptidases is
known to break down the proteins that keep the endothelial cells intact, therefore the
hypothesis is that inhibition of the family results in arteriogenesis and increased blood
flow. Increased MMP activity is assumed to lead to tipping the balance towards a pro-
proteolytic environment causing a decrease in mature collaterals, while increased TIMP
expression will cause decreased MMP activity, and henceforth lead to an anti-
proteolytic environment where there is an increase in mature collaterals. Indeed, it has
already been published that cerebral arteriogenesis induced by stroke causes an up-
regulation of TIMP1 in collaterals31,33. Outcomes from our canine work support this
claim following 10 weeks of prophylactic TCT supplementation. The current work seeks
to determine earlier changes in pro-arteriogenic target gene expression, including
TIMP1 and the aforementioned targets listed in Table 1. Briefly, mice were
supplemented prophylactically with TCT or vehicle placebo for 4-6 weeks prior to
mechanical occlusion of the middle cerebral artery to cause ischemic stroke in primary
somatosensory cortex. We describe a new technique to label collaterals during
ischemia and leverage experience with laser capture microdissection to selectively
measure gene expression of arteriogenic targets in collected perfused collaterals.
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Table 1. Primary literature reviews on arteriogenic factors
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Methods:
Animal Supplementation. All experiments were approved by the Institutional Animal
Care and Use Committee of the Ohio State University. Upon arrival, twelve C57BL/6 (5-
week old, Harlan, Indianapolis, USA) mice were allowed to acclimate to their home cage
environment for 1 week prior to supplementation. At the start of supplementation, mice
were randomized to one of two groups (n=6). Group one received supplementation for 4
weeks and group two received supplementation for 6 weeks. Mice were maintained
under standard vivarium conditions (22±2°C with 12:12 dark: light hour cycles). Within
groups, mice were evenly divided to receive placebo control (n=3) or TCT (n=3)
supplementation. The placebo group was orally gavaged five days a week with vitamin
E stripped corn oil using a volume matched to the mean of TCT supplemented mice
(Figure 5). The test group was orally gavaged with TCT (Carotech, Malaysia) in vitamin
E-stripped corn oil at a dosage of 50 mg/kg body weight as reported previously29.
Surgery was performed at 24h after the last supplementation.
Surgery. Acute ischemic stroke was induced using the intraluminal suture method of
middle cerebral artery occlusion (MCAO) as described previously29 (Figure 6).
Transient focal cerebral ischemia was induced in mice following 4 or 6 weeks of
supplementation. The mice were anesthetized with 1 to 1.5% isoflurane delivered by
medical air (21% O2, balance N2). A monofilament nylon suture (6-0) was advanced into
the internal carotid artery by way of the external carotid artery. The filament tip was
directed until slight resistance was felt (∼8mm). Cortical blood flow was continuously
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monitored during surgery using laser Doppler flowmetry (LDF). Successful MCAO was
confirmed by a >70% reduction in relative blood flow in the MCA supplied cortex. Once
MCAO was validated, the mice remained under anesthesia for 30min until FITC-lectin
perfusion was performed while MCAO-induced ischemia persisted.
Validation of acute ischemic stroke surgical model. Acute ischemic stroke is caused
by an abrupt and sudden occlusion of a large vessel which arrests cerebral blood flow
and the delivery of blood borne nutrients including oxygen and glucose to brain tissue.
To model this phenomenon in vivo, we employed the intraluminal suture method of
middle cerebral artery occlusion (MCAO, as depicted in Figure 6 of methods). This
approach induces ischemic stroke by mechanical occlusion of the MCA using a 6-0
nylon filament suture29. As real-time cerebral angiography is not enabled in our rodent
model, we rely on laser Doppler flowmetry (LDF) to validate successful MCA occlusion.
To acquire LDF from the occluded MCA territory, a 5mm incision is made in the scalp
for intracranial LDF probe placement. The probe is placed in the primary somatosensory
(S1) cortex using a small screw affixed with cyanoacrylate. LDF produces continuous
light (785nm), and gives measurements in arbitrary perfusion units (pfu). In order to
determine the decrease in blood flow using LDF, before the suture is inserted into the
middle cerebral artery, cortical blood flow is monitored to establish a pre-stroke
baseline. Once MCAO is successfully performed, blood flow is continuously monitored,
and successful occlusion is confirmed by a >70% reduction in relative blood flow as
compared to baseline. The table shown gives an exact percentage decrease in cortical
blood flow seen by each animal during surgery, conferring successful stroke in each.
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FITC-Perfusion. FITC-conjugated lectin (tomato Lycopersicon esculentum, Vector
Laboratories) was perfused during ischemia to visualize perfused collaterals of the
stroke-affected S1 cortex for use with laser capture microdissection experiments and
downstream molecular study. Mice remained anesthetized with isoflurane (1%–1.5%) in
medical air and FITC-lectin perfusion was performed while the MCA remained occluded.
Delivery of FITC-lectin particles during cerebral ischemia was achieved by intracardial
injection (250μL FITC-lectin) 30mins after the onset of MCAO. FITC-lectin was allowed
to circulate systemically for 5min after which deeply anesthetized mice were decapitated
and brain tissue collected and embedded in OCT (Sakura, Torrance, CA, USA).
Cerebellum brain tissue was snap frozen in liquid nitrogen for downstream HPLC
determination of vitamin E content.
Vitamin E Extraction and Analysis Using High Performance Liquid
Chromatography. Vitamin E extraction (Table 2) and analysis of mouse brain tissue
was performed as previously described using an HPLC-coulometric electrode array
detector (Coularray Detector, 12-channel, model 5600, ESA, Chelmsford, MA, USA)
(Figure 7). This system enables the simultaneous detection of five naturally occurring
vitamin E family members in a single run34 (Figure 8), including alpha-tocopherol,
gamma-tocopherol, alpha-tocotrienol, gamma-tocotrienol, and delta-tocotrienol.
Immunohistochemistry and imaging. At the time of euthanasia, mouse cerebral
hemispheres were sliced in the coronal axis using a brain matrix (Ted Pella, Inc.) and
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embedded in OCT (Figure 9). OCT-embedded frozen brain tissue was sectioned
(10μm) using a cryostat (CM3050s, Leica Microsystems, Buffalo Grove, IL, USA) and
mounted on positive-charged slides. Immunohistochemical staining of sections was
performed as described35 using CD31 (1:200; BD Pharmingen, San Diego, CA, USA)
primary antibody to label all blood vessels in the stroke-affected hemisphere regardless
of whether they were perfused. Secondary antibody detection and counterstaining were
performed as described previously35. The entire stroke-affected hemisphere was
imaged using fluorescent microscopy36. Perfused collaterals were quantified from FITC-
lectin labeled vessels and expressed as percent area of the entire hemisphere. Total
blood vessels in the stroke-affected hemisphere were quantified from all CD31 labeled
blood vessels.
Laser Microdissection Pressure Catapulting and RNA isolation. For laser capture
microdissection, OCT-embedded brain tissue was sliced into 12µm thick sections using
a cryostat. Sections were mounted onto RNase inhibitor-treated thermoplastic
(polyethylene napthalate)-covered glass slides (PALM Technologies, Bernried,
Germany). In this work we refer to collaterals as FITC-lectin perfused vessels isolated
from stroke-affected primary somatosensory (S1) cortex. FITC-lectin labeled collaterals
from the stroke-affected primary somatosensory cortex were collected using a PALM
MicroLaser, MicroBeam, and RoboStage/RoboMover system (Figure 10). More than
150,000µm2 of capture elements were collected (Table 3) for downstream RNA
isolation, cDNA synthesis and real-time PCR. After laser cutting, the isolated collaterals
were catapulted directly into 35μl of RNA extraction buffer (PicoPure RNA Isolation kit;
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Life Technologies, Grand Island, NY, USA) situated directly above the section in a
microtube cap. An additional 15μl of extraction buffer was added after collection. RNA
was isolated from captured and catapulted elements using the PicoPure RNA Isolation
Kit as described37.
Real-Time PCR. Gene expression levels of arteriogenic targets were independently
determined at 24 hours from contralateral control and stroke-affected laser
microdissection pressure catapulting-captured elements using real-time PCR, as
previously described37. Total RNA was reverse transcribed into cDNA using oligo-dT
primer and Superscript III (Life Technologies, Grand Island, NY, USA). Reverse
transcriptase-generated DNA was quantified by real-time PCR assay using double
stranded DNA-binding dye SYBR Green-I (Life Technologies, Grand Island, NY, USA)
with custom made primers (Integrated DNA Technologies, Coralville, Iowa, USA). All
primer sequences have been provided (Table 4).
Statistical Analysis. Statistical analysis of data was performed using Microsoft Excel
software (v12.3.6). All data are reported as mean ± standard deviation. Difference
between means was tested using Student's t-test (p<0.05).
A timeline is provided to clarify the chronological steps of the study (Figure 11).
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Results:
Verification of results in a rodent model. Informed by our canine study, we repeated
10 week supplementation studies in mice to specifically look at cerebral blood flow in
the brain during ischemic stroke. As in canine, 10 week prophylactic supplementation of
TCT in mice significantly attenuated stroke induced lesion volume (Figure 12).
Collateral blood flow was studied in these mice using laser speckle flowmetry imaging.
Results show that 10 week TCT supplementation is successful at increasing cerebral
blood flow to the stroke-affected region (Figure 13). On the basis of these outcomes,
the current work was designed to test expression of pro-arteriogenic factors at earlier
time points (4-6 weeks) to determine whether the TCT-mediated change in
arteriogenesis was an acute response (at the time of MCAO) or the result of chronic
changes over the course of supplementation.
Brain vitamin E levels following prophylactic TCT supplementation. Given the
natural variation in mouse development and body weight, mice were weighed weekly
and TCT gavage dose was adjusted accordingly to achieve a concentration of 50mg/kg
body weight. To account for variability in the amount of brain tissue collected at
euthanasia, vitamin E content was normalized to brain protein content (pmol vit E/mg
protein). Protein quantification was performed using the bicinchoninic acid assay (BCA,
Figure 14). The BCA protein assay is a biochemical assay used to determine total
protein concentration. The assay is colorimetric, meaning it relies on the detection of
light absorbance at 562nm in order to determine protein concentration by comparison to
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a standard. This technique was used to determine brain tissue protein content for each
mouse. Subsequently, vitamin E levels determined by HPLC were normalized to brain
protein concentration. Figure 15 demonstrates that TCT supplementation enriched
brain tissue with α-tocotrienol by 12.09 nmol/g protein. The murine diet provided is
deficient in all tocotrienols, resulting in the detection of no significant amount of
tocotrienol in brain tissue of the PBO controls using HPLC-coulometric electrode array
detection. Of note, the concentration of brain α-tocotrienol in TCT-supplemented
animals was 12.3 times less than that of α-tocopherol in the cerebellum, which
represents the most bioavailable vitamin E isoform found in the body (Figure 15). This
outcome is in line with known uptake mechanisms for alpha-tocopherol by the
tocopherol transfer protein (TTP), which has an 8.5-fold greater affinity for tocopherols
versus tocotrienols38, though this may vary based upon tissue type39,40. TCT
supplementation also modestly increased the concentration of α-tocopherol in brain
tissue as compared with PBO controls. Our TCT supplement, while highly enriched with
alpha-tocotrienol, is only 70% pure, the remainder of which contains other vitamin E
isoforms, including alpha-tocopherol. Taken together, results demonstrate that mice
orally supplemented with TCT for only 4-6 weeks significantly increased the
concentration of tocotrienols in the brain as compared to PBO controls.
Tocotrienol supplementation increases collateral perfusion during acute ischemic
stroke. Our recently published observation31 that prophylactic TCT supplementation
improved cerebrovascular collateral circulation during stroke uncovered new putative
mechanisms for TCT protection against stroke injury. This observation was enabled by
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a large animal (canine) endovascular stroke model that employed real-time cerebral
angiograms for documentation of cerebral perfusion during stroke. The canine study
revealed, through the retrospective study of angiograms, that collateral blood flow had
improved in the TCT supplemented canines. Angiograms revealed improved collateral
blood flow, allowing for retrograde filling of occluded parent vessels through
anastomosing collaterals (Figure 16). To confirm results, a physician was shown the
angiograms to objectively assess the collateral blood flow seen in each subject on an
11-point scale of the stroke-affected hemisphere. The physician, blind to the study, gave
significantly higher scores to the supplemented group when compared to the placebo.
This increased collateral blood flow is thought to be the reason for decreased infarct
volumes seen within the canine study. Until this work, TCT had been described to
attenuate stroke-induced injury on the basis of neuroprotection alone29. In light of cost,
molecular reagent availability, and low-throughput surgeries associated with the canine
stroke model, we sought to develop tools to test mechanistic hypotheses of TCT-
induced arteriogenesis in a small animal (murine) stroke model. To visualize cerebral
perfusion in mouse during MCAO, fluoroscopic angiograms are not a viable option. We
therefore developed the following immunohistochemical approach to visualize cerebral
perfusion during acute ischemic stroke. During ischemia, mice were injected with FITC-
conjugated lectin, sections of the same brain were also immunostained using CD31, a
marker of endothelial cells41. This lectin compound, a carbohydrate binding protein
aiding in molecular recognition specific to sugar moieties, is derived from tomato. The
name lectin itself is derived from the Latin term legere, which means “to select”. The
particular lectin used here binds selectively to perfused endothelial cells, allowing
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visualization of structures in the microvasculature when viewed by fluorescent
microscopy, refer to Figure 10. To stain all endothelial cells in brain tissue, regardless
of perfusion, we employed CD31 immunostaining. CD31 is an immunoglobulin
superfamily member. As an immonglobulin, CD31 mediates adhesion and trans-
endothelial migration of T-lymphocytes into the vascular wall, as well as T cell activation
and angiogenesis. CD31 is localized on the surface of endothelial cells42. Using these
two staining methods in combination allows for relative quantification of total vasculature
and perfused vasculature during ischemia (Figure 17). These images are then
compared and merged in order to show the change in perfused collateral blood flow of
the stroke-affected region of the brain, but not an overall increase in number of total
blood vessels present in the brain, lending credence to the process of arteriogenesis.
Quantification showed that prophylactic TCT supplementation significantly increased
lectin perfusion in the S1 cortex by 2.3 fold (p=0.01) following 4+ weeks
supplementation.
Endothelial cell specific laser capture microdissection (LCM) from brain tissue. A
major obstacle with studying brain vasculature is the contamination of other cell types in
collected tissue, such as neuron or glial cells. To avoid such contamination in collected
tissue samples, a technique known as laser capture microdissection (LCM) was
employed that enables site and cell specific resolution of tissue biology. This approach
allowed for selective capture of blood vessels from brain tissue using an LCM
microscope guided by FITC-lectin tagged endothelial cells of perfused vessels. Laser
captured collection yield (Table 3) shows the area of collected tissue from each
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samples, separating the stroke affected and contralateral sides for separate analyses.
With these tissue samples collected, RNA isolation was then performed using the
PicoPure RNA Isolation kit (Arcturus, Mountain View, CA, USA). Initial analysis of the
RNA was determined using a NanoDrop spectrophotometer (NanoDrop Technologies,
Wilmington, DE). The machine provided nucleic acid concentrations, as well as purity
information in the form of 260/280 and 260/230 ratios to determine protein or other
contaminants presence in the sample (Table 5). The ratio of 260/280 gives the
absorption found at 260nm light and 280nm. The nucleic acids absorb light best at
260nm, while proteins and phenols, which are commonly used in RNA extraction,
absorb light strongly at 280nm. The same principles hold true for the 260/230 ratio, as
thiocynates, carbohydrates, some phenols, and other organic compounds best absorb
light at 230. While an acceptable 260/280 ratio is between 1.8-2.0, our values were
consistent with the collection range for laser capture microdissection of ~1.531.
Screening for TCT sensitive arteriogenic markers. Arteriogenesis refers to a positive
outward remodeling of pre-existing collateral arteries into larger vessels, which are able
to circumvent sites of occlusion and perfuse otherwise stroke-affected tissue33,43. To
determine whether TCT supplementation invoked molecular mechanisms of cerebral
arteriogenesis at an earlier (4-6 week) time point, endothelial cells from the stroke-
affected and contralateral control cerebral cortex were selectively isolated and prepared
for PCR analysis. With prepared tissue, PCR analysis was used to confirm collection of
endothelial cells, using von Willebrand factor (vWF) as a endothelial cell marker, glial
fibrillary acidic protein (GFAP) as a glial cell marker, and neurofilament heavy
40
polypeptide (NFH) as a marker for neurons8 (Figure 18). Of interest were multiple
markers previously indicated to play a key role in the process of arteriogenesis44.
Previously identified markers includes; matrix metalloproteinase family members
(MMP2, MMP9), tissue inhibitor of metalloproteinase family members (TIMP1, TIMP2),
chloride intracellular channel family members (CLIC1, CLIC4), Krüppel-like Factor 2
(KLF2), cluster of differentiation variant 3 (CD44v3), P300/CBP-association factor
(PCAF), and Shc1 sensitive targets (EphrinB2 and delta-like 1 and delta-like 4). For
quick reference of arteriogenic targets, refer to Table 1.
Matrix metalloproteinases (MMP) 2 and 9 have previously been identified as key
contributors within the process of arteriogenesis44. MMPs are a family of zinc-dependent
endopeptidases which are able to break down extracellular matrix45. MMP2 and MMP9
belong to a class of MMPs known as gelatinases, which are unique from the other
MMPs in their role as proteolytic enzymes. This class is capable of breaking down
gelatin and type IV collagen found in the basal lamina, a layer of extracellular matrix
secreted by the endothelial cells. Due to this activity, the role of gelatinases is critical for
the remodeling of vessels. Our data shows this family is responsive to TCT
supplementation, with MMP2 mRNA being down-regulated significantly (2.46x
decrease, p=0.002, Figure 19), and MMP9 showing a trend towards down-regulation as
well (2.51x decrease, p=0.68, Figure 20), though the results were not statistically
significant.
41
Of specific interest during our project were TIMP1 and TIMP2, for their previously
identified roles as inhibitors of MMPs, aiding in the process of collateral remodeling via
arteriogenesis33. These genes and their products fall within a family known as tissue
inhibitor of metalloproteinase (TIMP). This family of genes encode for proteins of the
same name, which are natural inhibitors of the matrix metalloproteinases (MMPs).
TIMP1 and TIMP2 were chosen for study, as they are known inhibitors of MMP2 and
MMP9. With their ability to function as inhibitors of these MMP peptidases, the TIMP
family is known to play an important role as the regulator of MMPs in the brain. Previous
work has shown that TIMP1 gene regulation is responsive to TCT supplementation31.
Our findings here report a down-regulation of TIMP1 in the collected tissue samples
(2.49x decrease, p=0.01, Figure 21), which confirm sensitivity to supplementation, while
TIMP2 was not significantly affected (1.67x increase, p=0.17, Figure 22).
Previous work identified chloride intracellular channel (CLIC) 1 and 4 to be up regulated
by supplementation of TCT31. These CLICs belong to a group of genes and their protein
products that regulate processes including stabilization of cell membrane potential,
transport across the epithelium, cell division, apoptosis, and cell motility46,47. The CLIC
proteins are thought to influence the process of arteriogenesis by acting upstream of
other genes relevant for vascular growth, including hypoxia-inducible factor-1α, vascular
endothelial growth factor-A (VEGF-A), and angiopoietin-248. CLIC1 and CLIC4 have
been shown to be sensitive to TCT supplementation31. The CLIC family is also known to
play an important role in the process of cell hallowing and widening, known as
tubulogenesis, which is critical for functional arteriogenesis49,50. Data collected in this
42
study showed no changes in mRNA expression of either CLIC1 (1.67x decrease,
p=0.22, Figure 23) or CLIC4 (1.58x increase, p=0.20, Figure 24) markers at the earlier
4-6 time point for collection.
Krüppel-like Factor 2 is a newly identified arteriogenic marker51, it is understood to be
induced by shear-stress, resulting from laminar flow of blood through the vascular
tissue. The increased gene expression within the endothelial cells leads to anti-
inflammatory response due to inhibiting activation of p65, anti-thrombolytic effect
caused by up-regulation of endothelial nitric oxide synthase and thrombomodulin, and
anti-angiogenesis properties via inhibition of vascular endothelial growth factor (VEGF)
receptor 252. It is understood to be crucial in vascular development, with previous
research showing that knockout mice die around embryonic day 13 due to severe
embryonic hemorrhaging53,54, and thus is thought to be critical in vessel development
and integrity throughout life. Importantly, this work is the first to determine whether KLF2
mRNA expression is TCT sensitive. Results of this study have shown that TCT
supplementation causes a trend of moderate down-regulation in KLF2 that is very close
to statistical significance (3.29x decrease, p=0.08, Figure 25). We hypothesize that this
down-regulation would be significant if supplementation persisted to the 10 week time
point. Statistical significance may be reached at the 4-6 week time point if the study
were better powered.
Previous work has indicated that during the process of arteriogenesis, both total CD44
43
as well as CD44 variant 3 (CD44v3) are up regulated and result in improved
arteriogenic outcomes55. The gene encodes for a cell-surface glycoprotein, specifically
involved in cell-to-cell interactions, cell adhesion and migration. The protein functions as
a receptor to hyaluronic acid and other ligands, such as osteopontin, collagens, and
MMPs. This interaction with the MMP family is hypothesized as the connection between
these surface proteins and the process of arteriogenesis. CD44v3 is one of many
variants that arise from the transcription of this gene as transcripts undergo complex
alternative splicing that results in many functionally distinct isoforms56. This glycoprotein
has not been studied in previous TCT supplementation projects, and as such the gene’s
sensitivity to TCT was unknown. Though screened here, no data resulted from the
testing (Figure 26). We conclude that expression was either very low, or that
amplification did not result due to poor primer design. Further testing with new primer
design is required.
K (lysine) acetyltransferase 2B (KAT2B), also known as P300/CBP-associated factor
(PCAF), is a transcriptional co-activator associated with the p53 protein. This protein
has recently been indicated as playing a large role in arteriogenesis57. PCAF has
histone acetylating activity and promotes transcription of multiple inflammatory genes.
As arteriogenesis is an inflammatory driven process, PCAF is seen as a multi-factorial
regulator of this process through its regulation of inflammation. To confirm, recent
studies have shown that in mice, PCAF deficiency reduced the in vitro inflammatory
response in leukocytes and vascular cells involved in arteriogenesis interrupting the
process significantly. We had not previously study this gene, so it’s sensitivity to
44
supplementation is unknown. Results showed no alteration in the expression of PCAF
mRNA within the samples (p=0.86, Figure 27).
Shc1 is a gene that holds an important function in the process of arteriogenesis. The
gene is activated by the onset of shear stress and integral in two pathways necessary
for the arteriogenic process to be successful58. Shc1 knockout mice have impaired
activation of the nuclear factor κ-light-chain-enhancer of activated B-cell (NF-κB)-
dependent inflammatory pathway59. Additionally, Shc1 is required for shear-stress
mediated arteriogenic remodeling and induces up-regulation of the endothelial cell
marker ephrinB2 and activation of the Notch pathway by ligands delta-like 1 (DLL1) and
delta-like 4 (DLL4). To test whether targets of the Shc1 pathway were sensitive to TCT
supplementation, we assessed gene expression of EphrinB2, DLL1, and DLL4.
EphrinB2 is a target gene of the notch pathways, and was studied to identify a change
in notch pathway activity. EphrinB2 was shown to significantly decrease in mRNA
expression due to TCT supplementation (1.72x decrease, p=0.01, Figure 28). To
further this pathway, ligands of the notch pathway were also analyzed. These ligands
include delta-like 1 and delta-like 4. In these ligands, we saw no significant change and
an increase respectively. Results show delta-like 1 change being statistically
insignificant (6.44x increase, p=0.19, Figure 29), while delta-like 4 was significant
(3.96x increase, p=0.04, Figure 30). The genetic markers studied here have all been
consolidated into a schematic diagram to better represent their role in the process of
arteriogenesis (Figure 31).
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
Discussion:
The motivation for ischemic stroke research is driven by the paucity of clinically
available therapies for stroke patients. While a number of stroke therapies have shown
promise in pre-clinical testing, few have proven effective in a clinical setting60. A recent
review of 8,516 stroke related studies identified 1,026 candidates in pre-clinical testing
that showed promise for treatment of ischemic stroke61. To date, each of these have
fallen short in clinical trials62. Due to this grave need for therapeutic options to treat
ischemic stroke patients, we set about testing TCT against ischemic stroke. Our lab has
been studying tocotrienol vitamin E isoforms in the context of neuroprotection against
ischemic stroke for over a decade11,21,29,31,37,63-73. As a natural vitamin, humans have
safely consumed tocotrienols as part of a complex diet for centuries74,75. The most
abundant natural source of TCT vitamin E comes from palm oil. Palm oil is
predominantly produced in Malaysia and Indonesia, which account for around 85% of
the world’s palm oil76. As the world’s principal producer of palm oil, the palm oil plant is
significant to the agriculture and economy of both Maylasia and Indonesia. In Malaysia
the palm oil industry accounts for 3.2% of the national GDP and 6 to 7% of the national
GDP in Indonesia77. Recently, palm oil derived tocotrienol vitamin E has been granted
Generally Recognized As Safe (GRN No. 307) certification by the United States Food
and Drug Administration (U.S. FDA) for use as a food ingredient78. In light of a robust
safety profile and growing body of research testing mechanisms of both neuro- and
vascular-protection, we have begun clinical testing of tocotrienol vitamin E in patients
who already suffered a sentinel ischemic stroke (see NCT01578629 on
67
clinicaltrials.gov). This patient population is at high risk for a second stroke event within
one year of their first stroke episode, therefore serving as an ideal population of study
for a prophylactic intervention such as TCT. This work, returns to pre-clinical testing to
elucidate mechanisms of the vascular protective properties of TCT against acute
ischemic stroke. Outcomes may direct future clinical trial designs that specifically target
vascular protective intervention, such as catheter based delivery or long-term
prophylactic dietary supplementation.
In light of the failure for putative stroke therapeutics that found success in rodent pre-
clinical testing but failed in human clinical trials, we sought to test TCT against ischemic
stroke in a large animal model that more closely approximated the human stroke
condition. The mongrel canine stroke model required two small femoral artery
punctures, followed by insertion of a platinum embolic coil to occlude the middle
cerebral artery, as confirmed by c-arm fluoroscopy79. This testing showed that TCT
does attenuate stroke-induced injury in a canine brain that more closely approximates
that of a human. This study also uncovered an important clue into a previously unknown
mechanism for TCT protection against stroke-induced brain injury. Specifically, cerebral
angiograms enabled in our large animal setting demonstrated that TCT but not placebo
treated canines possessed increased collateral blood flow in the stroke hemisphere.
This observation was the first to identify a TCT-dependent vascular protection
mechanism against stroke-induced injury31. As no new vessels were seen within the
angiogram, the protection was understood to take place via remodeling of pre-existing
collaterals, a process referred to as arteriogenesis. On the basis of this observation, we
68
move back to the mouse in order to more easily study the mechanisms of collateral
remodeling identified in our large animal, canine stroke model. The mouse is better
suited for this study because of decreased cost associated with the model, increased
availability of test subjects, and access to molecular biology reagents for testing
purposes.
Our first step when moving to a mouse model was to validate the improvement in
collaterals observed in canine testing. To do so, an identical supplementation
experimental design was employed for mice. They were supplemented for 10 weeks,
then given stroke and compared to their placebo counterpart for both infarct volumes as
well as blood flow to the stroke-affected hemisphere. Real time angiography was not
feasible in mouse, as their smaller size limits fluoroscopy exposure. Therefore, in order
to verify an increase in blood flow, laser speckle contrast imaging was employed. Laser
speckle contrast imaging work by shining a laser on the surface of the cortex, causing
the light to scatter and produce a speckle pattern that can be captured by a camera.
Analysis of the speckle pattern leads to a so called “speckle contrast”, which is then
related to a measure of blood flow for each area of the cortex80,81. Results showed that
10 week prophylactic TCT significantly increased blood flow in the stroke-affected tissue
as compared to PBO supplemented controls. Furthermore, stroke-induced lesion
volume was attenuated as measured by 11.7T MRI. These outcomes were consistent
with results observed in our published canine work31 (Figures 30, 31).
69
From here we began to study the mechanistic basis for this arteriogenic
cerebrovascular remodeling we had seen in both the canine and mouse model. One of
the first questions that arose was the time frame of supplementation required for this
process to be effective. As we observed successful remodeling and improved collateral
circulation during stroke in mice supplemented with TCT at 10 weeks, we elected to
also study an earlier time frame of 4-6 weeks to detect earlier changes in gene
expression that may affect the long acting improvement in blood flow during stroke.
Originally, we had proposed to separate the two groups into separate time points and
compare the two, however there was no significant different in vascular changes seen
between the groups, so they were joined together as one earlier time point trial. By
doing this, we aimed to shed light on the temporal resolution of the arteriogenic
transcriptome, by comparing pro-arteriogenic markers studied at the 10 week time point
of the published canine study to the 4-6 week time point. In addition to the few genetic
markers studied in the 10 week study, we also identified new pro-arteriogenic
candidates for their sensitivity to TCT supplementation after 4 weeks.
Importantly, this is the first work to test vitamin E levels in brain as early as 4 weeks
after start of supplementation. Results revealed that the concentration of alpha-
tocotrienol in brain after one month of supplementation is sufficient to afford
neuroprotection on the basis of previously published work39,67. We hypothesize that this
amount is also adequate to induce vascular remodeling. As it relates to vascular
protection here, TCT supplementation significantly decreased TIMP1 expression in
perfused vessels of the stroke-affected primary somatosensory cortex. TIMP1’s ability
70
to control extracellular matrix degradation and advancing vascular remodeling by the
activation of cell proliferation represents an important role in arteriogenesis33. The
observation that this supplementation may decrease TIMP1 expression in the blood
vessel of stroke-affected hemisphere points to the possibility that oral supplementation
of TCT may prime the cerebral vasculature, enabling arteriogenesis in response to an
ischemic event in the cerebral cortex. Our understanding is that the onset of the
arteriogenic remodeling process is marked by an initial decrease in expression of the
protease inhibitor TIMP1. The targets of TIMP1, MMP2 and MMP9, belong to a class of
MMPs known as gelatinases, which are unique from the other MMPs in their role as
proteolytic enzymes. This class is capable of breaking down gelatin and type IV
collagen found in the basal lamina, a layer of extracellular matrix secreted by the
endothelial cells82,83. This process requires break down of the basal lamina to increase
vessel diameter. Proteolysis of the internal and external basal lamina by MMPs is
essential to overcome the structural barriers to growth84-87. The controlled breakdown of
this vascular framework allows for the expansion and outward growth of collateral
vessels32, necessary for remodeling. Though previous work has suggested that at 10
weeks TIMP1 is significantly increased31,33, our outcomes point to an acute phase
decrease in TIMP1 that allows for more MMP2 and MMP9 gelatinase activity. This early
response likely aids the remodeling process, while a late-phase (i.e. 10 weeks) increase
in TIMP1 would help stabilize the functional vessel and prevent further remodeling or
other break down leading to leakiness within the vessel. Overall, this work provides
further evidence of TCT supplementation regulating TIMP1 expression and
subsequently invoking cerebrovascular arteriogenesis.
71
In addition to TCT supplementation exerting influence over the proteolytic environment
during cerebrovascular remodeling, it also appears to affect shear stress and related
mechanotransduction signaling involved in the process of arteriogenesis. Previous work
identified Shc1 as a shear-stress sensitive inducer of arteriogenesis59. Phosphorylated
Shc1 adaptor protein is responsible for activating multiple tyrosine kinases, including
Ras and MAPK which are believed to induce transcriptional activation of Notch ligands
(i.e. Delta-like ligands 1 and 4)59,88,89. Shc1 is required for shear stress induced
activation of these targets59, so to indirectly study this adaptor protein we assessed
mRNA expression of mechanotrasnduction-sensitive notch ligands, including DLL1 and
DLL4. Interestingly, our results show DLL4 mRNA is significantly up-regulated (3.96x
increase) by TCT supplementation, while DLL1 shows a trend of increased expression
(6.44x increase, p=0.194). It would be expected that an increase in DLL1 and DLL4
mRNA expression would drive a concomitant increase in ephrinB2 expression. Here, no
such effect was observed, suggesting the following possible explanations: (1) the time
point for collection preceded DLL1 and DLL4 dependent induction of ephrinB2 in the
notch signaling pathway, (2) post-transcriptional silencing of ephrinB2 by unknown
mechanisms, (3) post-translational degradation of DLL1 and DLL4 protein, or (4)
interference of DLL1/DLL4 notch ligand binding. Recent research on DLL1 and DLL4
show they have separate function. Pertinent literature shows that DLL1 is essential for
proper function of shear stress induced arteriogenesis in the post-natal mouse90, while
DLL4 is only expressed in microvessels of the post-natal mouse, or all vessels during
embryonic development91. In this light, TCT-dependent induction of DLL4 suggests two
72
possibilities: (1) that the majority of laser captured elements were smaller microvessels
(capillaries, Ø<10µm Æ arterioles, Ø=10-50µm), or (2) that TCT supplementation is
leading to a “reprogramming” of endothelial cells, awakening normally dormant
embryonic development genes such as DLL4. Overall, we hypothesize that an increase
in Shc1 expression has occurred, due to the up-regulation of its closest studied
downstream targets, DLL1 and DLL4. On the basis of these results, there is a clear
need to study molecular target upstream of the delta-like ligands, specifically Shc1. To
gain a better understanding of the role TCT supplementations is playing here, analysis
of Shc1 mRNA and protein is required. To do so, Shc1 mRNA expression can be tested
using real-time quantitative-PCR, protein expression can be tested using either Western
Blot techniques or immunohistochemical staining, and finally protein activity can be
determined by stoichiometric quantification of phosphorylated enzyme (active)
compared to un-phosphylated enzyme (inactive)92. Due to the findings here, future
experiments will address the notch signaling pathway in greater detail to further uncover
the role it plays in shear stress induced arteriogenesis.
In addition to DLL1, DLL4, and ephrinB2, we investigated another pro-arteriogenic
target known to be induced by shear stress and mechanotransduction signaling. KLF2
acts as an important transcriptional regulator of many processes within the body
including cell proliferation, apoptosis, differentiation and development93,94. Relevant to
this study, KLF2 is known as a shear stress-induced transcription factor capable of
activation many proteins95. Critically, these proteins have anti-inflammatory as well as
anti-coagulant properties. KLF2 is able to respond to shear stress by binding of
73
transcription factors to its promoter region. Many of the transcriptional factors are
components of the shear stress regulatory complex, such as the coactivator PCAF96.
Binding of these factors leads to the increased expression of KLF2 due to the increase
in shear stress associated with ischemia97,98. Confirming this, work has been done to
show that indeed a significant up-regulation of KLF2 is seen when high stress
environments are applied for long periods of time99. The downward trend in expression
of the KLF2 mRNA seen here indicated a decrease in shear stress has occurred. This
suggests that 4 weeks of supplementation is sufficient to cause arteriogenic remodeling,
resulting in an increase in vessel diameter and therefore a decrease in shear stress.
Interestingly, findings have highlighted KLF2’s ability to regulate hypoxia inducible factor
1-alpha (HIF-1α), as KLF2 promotes the degradation of HIF-1α100. Research on HIF-1α
has shown that at an early time point, such as 30 minutes, increased HIF-1α expression
is neuroprotective101. This additional mode of neuroprotection gives possible
explanation to the decrease in KLF2 mRNA seen here. To test shear stress directly we
plan to continue ongoing research using ultrasound to measure blood flow and
corrosion cast to find diameter of the vessels, allowing for quantification of shear stress.
The trend was very close to significance, and we believe that additional future additional
test subject would be enough to make this statistic significant. Lastly targeted for its role
in arteriogenesis through shear stress is PCAF. This transcriptional coactivator acts as
a master switch equipped with histone acetyltransferase activity57. PCAF can acetylate
both histone proteins and modulate nonhistone proteins, including those of the notch
pathway mentioned previously102. Results here show a lack of response to TCT
supplementation.
74
Another group of markers tested in this study were the chloride intracellular channel
(CLIC) genes. Our previous research in a canine model has also indicated that CLICs
are sensitive to TCT supplementation. There is also research to show CLICs role in
collateral reformation31,103. These Chloride intracellular channel proteins are required for
endothelial cell hollowing50, which is essential for the vascular remodeling process. With
this, we tested both CLIC1 and CLIC4 in the 4-week supplementation study. Our current
work revealed no change in mRNA expression for either of the CLIC markers.
The current work leverages laboratory expertise with complex surgical models (rodent
MCAO) and cutting edge technologies for cell and site specific resolution of tissue
biology (Laser Capture Microdissection). While these approaches strengthen the work
overall, they are not without their own limitations which are considered here. First and
foremost, our surgical stroke model possesses strengths and weaknesses, which have
been widely studied104-107. An advantage of the intraluminal suture model of middle
cerebral artery occlusion is that it demonstrates a very good representation of ischemic
stroke in nature. The method allows the restoration of blood flow after the induction of
ischemia, which mimics the series of events in human stroke and allows for assessment
of reperfusion108, though this is not utilized in our project, as we euthanized during
ischemia. Additionally, this is the most widely used stroke model in research, allowing
for widespread comparison to other stroke research109. Limitations of the model include
variability of infarct size and location, high mortality rate, hemmorraging107, and all
75
complications that may result from a surgical procedure such as infection,
immunodepression110,111, hyperthermia112,113, and loss of body weight114,115. Despite all
of these limitations, the MCAO stroke model is still the most widely used and
reproducible rodent model for acute ischemic stroke research. Of note, our laboratory
takes additional measures to improve reproducibility and mitigate some of the
aforementioned limitations. The use of laser doppler flowmetry is utilized to measure
decrease in blood flow. This allows for increased consistency in the size and location of
stroke.
Our use of laser capture microdissection also warrants consideration. One benefit of
this technique in relation to our study, is that it allows for selective isolation of
endothelial tissue. Without this, we would be forced to use whole tissue, and have to
deal with much larger contamination of samples by non-specific glial and neuronal cells.
Overall, this selective extraction of desired cells from heterogeneous tissues allows for
improved subsequent molecular analyses116. As this technique allows for increased
selectivity in cell type, it results in increased specificity of isolated RNA for downstream
testing117. This technique also comes with inherent downside. One such negative is
degradation of RNA due to slide handling and procurement procedures. As necessitated
by our protocol, slides and tissue are exposed to solutions required for dehydration,
staining, and other preparations as outlined in materials and methods. All of these steps
pose increased risk and time of exposure to RNase activity. To combat these risks, we
treat slides with RNaseZap (Ambion, Austin, TX, USA) before sectioning, and used
RNAlater (Ambion, Austin, TX, USA) once sections are mounted on the slides. Both
76
these techniques aid in the inactivation of RNase activity, aiding in the stabilization of
the RNA. The entire LCM process requires a large amount of time while on the
microscope, subjecting samples to greater risk of RNA degradation. In recognition of
this limitation, for the current study we limited collection of endothelial cells to 30min per
slide. Another problem is the amount of tissue collected. As the technique calls for
selection of only specific cell types, the amount of tissue collected for downstream
testing is vastly decreased as compared to ground whole tissue preparations. The
decrease in tissue quantity is mirrored by lower RNA yields, leading to difficulties with
further analysis of gene expression. To minimize these issues we optimized our protocol
for collection for up to 2.1*10^5 µm2. We also sought to increase the amount of tissue
collected by increasing section thickness to 12 µm. These strategies allowed for
successful yields of RNA necessary for downstream testing.
Our novel immunohistological approach using CD31 and FITC-lectin staining of
analyzing for quantification of stroke must also be considered. The approach enables
visualization of perfused vessels in the stroke-affected hemisphere along with direct
comparison to the contralateral hemisphere. Through immunohistochemical analyses,
this approach provides quantitative analyses of collateral improvement in the stroke-
affected hemisphere. On the other hand, these staining techniques are not always
accurate, and it is possible that not every vessel was stained. Contributing to this, there
is a varied amount of endothelial cell markers present in the vascular bed, and this is
constantly in fluctuation due to a combination of factors, including vessel size,
anatomical compartment differences within organ, and presence of pathological
77
conditions118. In addition to the staining of vessels, the software program that analyzes
the stains has limitations. The program uses automated selection and quantification
processes that can over- or under-estimate vessels that can be seen by the user, or
mark background stain that are not indicative of a stained vessel as intended. To
combat this the mosaic images used on the scope have been adjusted to maximize the
software’s recognition of stained regions.
Now with our insight into the outcomes from mRNA gene expression analyses, we have
new testable hypotheses and strong starting points for additional studies to be
conducted in the future. First we plan to look at activity of pro-arteriogenic enzymes
identified here and listed throughout this discussion. An understanding of enzyme
activity will help shed greater light on the how it is that TCT is able to induce
arteriogenic remodeling of cerebrovasculature. These future studies hold grave
importance in both the protease and shear stress pathways studied within this research.
Secondly, use of corrosion casts to directly measure vessel growth and diameter hold
great promise. With this information, as well as blood flow through larger vessels as
measured by ultrasound, we could calculate shear stress, and directly measure its
change throughout a study to see its overall effect. Finally, we will continue to study the
role played by TIMP1 in both TCT supplementation and arteriogenesis. We plan to
utilize TIMP1 knockout mice in order to test the significance of TIMP1 on stroke related
damage. Of further interest is whether TCT supplementation in TIMP1 knockout still
attenuates stroke-induced brain injury. If not, outcomes will suggest that TIMP1 is
essential for TCT-mediated protection against stroke. Overall, the project provides new
78
insight into mechanisms of arteriogenesis in response to TCT supplementation, and
gives direction to a multitude of future research projects.
79
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